Cell migration is present in virtually all life processes, including fertilization, embryogenic development, immune response, wound healing, and tumor metastasis. To improve the treatment of diseases associated with these various life processes, it is important to understand the underlying mechanisms of cell migration involved. This often requires that we recreate the environment that leads to and supports the continuous migration of cells. Here, we present two engineering approaches toward such a goal, with the additional emphasis that cell migration can be conducted in the absence of fluid flow, a mechanical stimulus that is known to influence cell behaviors. We chose the primary human neutrophil, which is highly motile and sensitive to both fluid flow and chemoattraction, as the model cell type for all our studies.
In the first approach, we used fluid flow to create a linear and time-invariant gradient of chemoattractants to guide the migration of neutrophils. A thin and porous membrane was used to screen off the associated flow forces while still permitting the diffusion of the gradient to the neutrophils. We showed that the membrane-based system is capable of directing neutrophil migration without the bias from fluid flow, and allowed within minutes the exchange of media to label and wash the migrated neutrophils. To assess the reduction of flow forces enabled by the membrane, we developed an analytical model to predict the direction and the magnitude of flow within the system. The validity of the model was verified both experimentally and numerically with particle tracking and computational fluid mechanic (CFM) simulations. We also performed total internal reflection fluorescence (TIRF) microscopy to verify the preservation of the gradient after v its diffusion through the membrane.
In the second approach, we created immobilized gradients of the chemoattractant interleukin 8 (IL-8) and the intercellular adhesion molecule 1 (ICAM-1) in the attempt to guide neutrophil migration. A gradient of soluble factors is first established, and the resulting difference of concentration over space leads to a bias in the binding of the soluble factors unto the substrate, forming an immobilized gradient. The immobilization is mediated by a combination of different physicochemical linkages, including electrostatic attraction, protein/protein interactions, and covalent bonding. We showed through labeling with fluorescent antibody that the number of IL-8 or ICAM-1 immobilized in a given area could be controlled, and varied over distances to form different gradient profiles. We further showed that our immobilization procedure does not affect the ability of IL-8 and ICAM-1 to activate and bind the neutrophils. However, with all the immobilized gradients that we have created so far, none were able to effectively promote the directed migration of neutrophils in long distances. Additional work is therefore required to establish if an immobilized gradient of either IL-8 or ICAM-1 alone can direct the migration of neutrophils in long distances, and if it does, what are the required conditions. Currently, our efforts suggest that the membrane-based chemotaxis system is a more attainable platform for promoting a directed migration that is shear-free.
The presented thesis work offers many potential applications. The membrane-based chemotaxis system, which has the general structure of two compartments separated by a membrane, resembled many physiological structures, including bone marrow, blood vessel, blood-brain barrier, hepatic portal vein, nephron in the kidneys, and alveolus in vi the lungs, and therefore serves as a versatile platform for understanding the transport phenomenon and the biochemical signaling in the aforementioned tissues. With improvements, the membrane-based system can also host larger-scale cell culture for protein production and tissue engineering. The protocols established for the gradient immobilization also provided many valuable references. These include: 1. A 1st order approximation of the reagents and the times required to fully saturate the substrate to be functionalized. 2. An automated image processing tool to measure the various parameters of cell motility. 3. A statistical framework to detect the presence of a directed migration. In theory, the standard operating procedures established are applicable to the surface functionalization with other peptides and proteins.
|Advisor:||Waugh, Richard E., McGrath, James L.|
|Commitee:||Brown, Edward, DeLouise, Lisa A., Kim, Minsoo|
|School:||University of Rochester|
|Department:||Hajim School of Engineering and Applied Sciences|
|School Location:||United States -- New York|
|Source:||DAI-B 76/08(E), Dissertation Abstracts International|
|Subjects:||Biomedical engineering, Immunology|
|Keywords:||Chemotaxis, Membrane, Microfluidics, Neutrophil|
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